JMML

Question 1

Estimated frequency of JMML?

Answer 1

Juvenile myelomonocytic leukemia (JMML) is an aggressive
myeloproliferative neoplasm (MPN) of childhood that occurs
with an estimated frequency of 1.2 cases per million

Question 2

How sensitive and specific is hypersensitivity of myeloid progenitor cells to granulocyte-macrophage colony stimulating factor (GMCSF) in colony-forming assays for JMML?

Answer 2

One of the hallmark laboratory features of JMML, hypersensitivity of myeloid progenitor
cells to granulocyte-macrophage colony stimulating factor (GMCSF) in colony-forming assays, can also be present in certain viral infections, thus providing a sensitive but nonspecific
adjunctive diagnostic tool.

Question 3

JMML. T or F: At presentation, a rare circulating
nucleated red blood cell can also usually be found in the
peripheral blood smear in JMML

Answer 3

True

Question 4

JMML. Around what proportion of JMML patients will have elevated HbF at presentation?

Answer 4

approximately 50% of patients will

present with an elevated hemoglobin F corrected for age

Question 5

JMML. Initiating genetic lesions in JMML predominantly affect genes encoding proteins in which pathway?

Answer 5

The spectrum of mutations described thus far in
JMML occur in genes that encode proteins that signal through the Ras/mitogen-activated protein kinase (MAPK)
pathways, thus providing potential new opportunities for both diagnosis and therapy. These genes include NF1, NRAS,
KRAS, PTPN11, and, most recently, CBL.

Question 6

Current diagnostic criteria for JMML?

Answer 6

The following criteria are required in order to diagnose JMML:[1]
All 3 of the following:
1. No Philadelphia chromosome or BCR/ABL fusion gene.
2. Peripheral blood monocytosis >1 x 109/L.
3. Less than 20% blasts (including promonocytes) in the blood and bone marrow (blast count is less than 2% on average)

Two or more of the following criteria:

1. Hemoglobin F increased for age.
2. Immature granulocytes and nucleated red cells in the peripheral blood.
3. White blood cell count >10 x 109/L.
4. Clonal chromosomal abnormality (e.g., monosomy 7).
5. Granulocyte macrophage colony-stimulating factor (GM-CSF) hypersensitivity of myeloid progenitors in vitro.

Question 7

Name some haematological manifestations of Noonan syndrome

Answer 7

Some children with Noonan syndrome also display a hematologic phenotype, including a self-resolving myeloproliferative disorder in infancy that resembles JMML, and bleeding diatheses. In 2001, Tartaglia and Gelb identified germ-line mutations in PTPN11, a gene encoding the non-receptor tyrosine phosphatase protein SHP-2, as causative in approximately 50% of children with Noonan syndrome. Based on the observation that children with Noonan syndrome can display myeloproliferative features, mutations in PTPN11 were subsequently identified to occur as somatic lesions in de novo, nonsyndromic JMML in as many as 35% of patients

Question 8

What is uniparental disomy? What is the difference between heterodisomy and isodisomy?

Answer 8

Uniparental disomy (UPD) occurs when a person receives two copies of a chromosome, or of part of a chromosome, from one parent and no copies from the other parent.[1] UPD can be the result of heterodisomy, in which a pair of non-identical chromosomes are inherited from one parent (an earlier stage meiosis I error) or isodisomy, in which a single chromosome from one parent is duplicated (a later stage meiosis II error).[2] Uniparental disomy may have clinical relevance for several reasons. For example, either isodisomy or heterodisomy can disrupt parent-specific genomic imprinting, resulting in imprinting disorders. Additionally, isodisomy leads to large blocks of homozygosity, which may lead to the uncovering of recessive genes, a similar phenomenon seen in children of consanguineous partners. Isodisomy is an example of copy neutral loss of heterozygosity. acquired uniparental isodisomy.
Relevant in cancer as it is a good way of ending up with two inactivating mutations in a tumour suppressor gene - this has been shown to occur in cases of
NF1-associated JMML.

Question 9

How would you screen for uniparental isodisomy?

Answer 9

high-density single-nucleotide polymorphism arrays

Question 10

What is cbl?

Answer 10

Cbl is an E3 ubiquitin ligase that is known to mark activated receptor and non-receptor tyrosine kinases and other proteins for degradation by ubiquitination, but also retains important adaptor functions

Question 11

Do JAK2 mutations occur in JMML?

Answer 11

No, unlike other MPNs of adulthood.

Question 12

What are the commonly occurring mutations in JMML? Which signalling pathway are they all involved in?

Answer 12

The current spectrum of commonly occurring
mutations in NF1, RAS, PTPN11, and CBL converge on the
Ras/MAPK pathway in a highly specific way that remains to be fully elucidated.

Question 13

True or false: most patients with Noonan

syndrome who present with a JMML-like MPN in the neonatal period will spontaneously resolve over the first year of life

Answer 13

True, somewhat akin to the transient myeloproliferative syndrome of Down syndrome (although Noonan syndrome-MPN is slower to resolve than Down syndrome-transient myeloproliferative disorder). Rare Noonan syndrome patients may require some low- or
intermediate-dose chemotherapy to improve splenomegaly and to control high white blood cell counts, but there is no longer the expectation that these children will require HSCT if they show clinical improvement in the first year of life.

Question 14

True or false: individuals with Noonan syndrome
who suffer from transient MPN of infancy are at an
increased risk of myeloid malignancies later in childhood

Answer 14

FALSE. Unlike patients with Down syndrome, individuals with Noonan syndrome who suffer from transient MPN of infancy do not appear to be at an increased risk of myeloid malignancies later in childhood.

Question 15

True or false: Some patients with Noonans develop a ‘self-resolving’ JMML? Signature characteristics of such cases?

Answer 15

True, though rare and difficult to predict. Among 12 patients observed without HSCT for more than 3 years from diagnosis, there were 5 patients experiencing long-term survival who harbored various RAS mutations.
Of great interest in both series is that spontaneously resolving patients all presented with favorable prognostic factors: young age at diagnosis, platelet counts above 33,000/uL, and relatively low age-adjusted hemoglobin F levels.

Question 16

JMML. Which clinical factors have been associated with a

poorer prognosis, and thus should sway toward earlier HSCT?

Answer 16

1. Older age at diagnosis (2 years),
2. Increased fetal hemoglobin for age (Hgb F), and/or
3. Platelet count under 33,000/uL at presentation, as well as
4. Female sex in some studies

These factors consistently correlate with a poor outcome
(reduced event-free survival [EFS] and overall survival [OS]) following allogeneic stem cell transplantation

Question 17

Is there any evidence that mutational status may be correlated with JMML clinical features and prognosis?

Answer 17

Limited. In 2005, Locatelli reported improved EFS of 64% with a 3-year median follow-up for 100 patients treated with allogeneic bone marrow transplant in the EWOG-MDS consortium. In that series, mutational status of NF1,
PTPN11, or RAS was not statistically significant as independent risk factors for survival. Yoshida et al. evaluated the clinical course and laboratory findings of 49 JMML patients, 32 of whom harbored mutations in NF1, KRAS, NRAS, or PTPN11. In their study,
mutation of PTPN11 was associated with older age at diagnosis (24 months), increased Hgb F (10%), reduced overall survival, and, importantly, appeared to be an unfavorable prognostic factor predicting relapse following transplantation.

Question 18

What is the evidence that gene expression signatures can help prognosticate in JMML?

Answer 18

Bresolin et al. reported that gene expression signatures
could segregate JMML patients into those who displayed an AML-type signature versus those who did not. These signatures were significantly associated with outcome, with those who displayed an AML-type signature experiencing a 10-year EFS of 6% and those without the AML-type signature who experience a 63% EFS. Of interest was the statistically significant correlation between an AML-type signature and known prognostic variables at diagnosis, including older age, lower platelet count, and higher Hgb F levels. However in a multivariate analysis, only the AML-type gene expression signature retained significance, although the levels of Hgb F and platelet count in this model were not defined at the same
levels as previously determined to carry prognostic significance.

Question 19

What does a farnesyltransferase inhibitor do?

Answer 19

The farnesyltransferase inhibitors (FTIs) are a class of experimental cancer drugs that target protein farnesyltransferase with the downstream effect of preventing the proper functioning of the Ras (protein). Studies have suggested that interference with certain post-translational modification processes seem to have quite a high selectivity for targeting cells displaying tumour phenotypes although the reason for this is a matter of controversy (as will be explained below).
After translation, Ras goes through four steps of modification: isoprenylation, proteolysis, methylation and palmitoylation. Isoprenylation involves the enzyme farnesyltransferase (FTase) transferring a farnesyl group from farnesyl pyrophosphate (FPP) to the pre-Ras protein. Also, a related enzyme geranylgeranyltransferase I (GGTase I) has the ability to transfer a geranylgeranyl group to K and N-Ras (the implications of this are discussed below). Farnesyl is necessary to attach Ras to the cell membrane. Without attachment to the cell membrane, Ras is not able to transfer signals from membrane receptors. Unfortunately, the predicted “early potential [of FTIs] has not been realised”.  The anti-tumour properties of FTIs were attributed to their action on Ras processing; however this assumption has now been questioned. Of the three members (H, N and K) of the Ras family, K-Ras is the form found most often mutated in cancer. As noted above, as well as modification by FFTase an alternative route to creation of biologically active Ras is through GGTase modification. When FFTase is blocked by FFTase inhibitors this pathway comes into operation – both K and N-Ras are able to be activated through this mechanism. In recognition of this a joint administration of FTIs and GTIs was tried, however this resulted in high toxicity. It is in fact thought that the lack of FTI toxicity may be due to a failure to fully inhibit Ras: FTIs actually target normal cells but alternative pathway allow these cells to survive. So how to explain the preclinical successes showing that many N- or K-Ras transformed cell lines (and even tumor cell lines that do not harbor Ras mutations) are sensitive to FTase inhibitors? It has been suggested that this is due to inhibition of farnesylation of a number of other proteins.[1] Therefore it is hoped that FTIs, whilst not Ras specific, still have potential for cancer therapy.

Question 20

JMML. Any evidence for FTI use?

Answer 20

Clinical responses to the farnesyl-transferase inhibitor were encouraging; however, there were no data to suggest that inhibition of Ras prenylation accompanied these responses. Indeed, the promise of using farnesyl-transferase inhibitors as “Ras” inhibitors is misleading,
because alternative geranylation can substitute for prenylation, a critical step in the activation of Ras.

Question 21

JMML treatment. What did the EWOG-MDS/EBMT JMML trial show for patients who received either no pre-HSCT therapy or low-dose chemotherapy pre-HSCT compared with high-dose acute myeloblastic leukemia-like chemotherapy?

Answer 21

They had identical EFS (52% vs. 50%), relapse incidence (35% vs. 38%), and treatment-related mortality (13% vs. 13%). Given the heterogeneity of disease burden at
presentation in children with JMML, it is not possible to be definitive about the patients who were given various pretreatment therapies and the impact of those treatments on ultimate outcome.

Question 22

What is the current stance on pre-transplant slenectomy in JMML?

Answer 22

Don’t do it. The previous COG trial for JMML mandated splenectomy for all children prior to HSCT. However, the EWOG-MDS/EBMT JMML trial demonstrated that comparing children with a spleen size 5 cm or 5 cm, children undergoing splenectomy had no statistical benefit in event-free survival (EFS; 61% vs. 44% vs. 48%,
respectively), relapse incidence (24% vs. 45% vs. 39%, respectively), or treatment-related mortality (15% vs. 11% vs. 13%, respectively). Therefore, neither splenectomy nor high-dose chemotherapy are recommended prior to a patient receiving conditioning for an allogeneic stem-cell transplantation unless clinically indicated for symptomatic relief.

Question 23

JMML treatment. Describe four ‘holding measures’ for patients awaiting an appropriate donor who have very high white blood cell counts, pulmonary problems,
and/or prominent organomegaly. How might the effectiveness of these treatments be monitored?

Answer 23

Oral 6-MP (50 mg/m2/d) with or without cis-retinoic acid (100 mg/m2/d). For severely ill children, or those children who progress on less-intensive therapies, low-dose intravenous cytarabine (40 mg/m2/d for 5 d) can be administered. If this fails, the combination of high-dose cytarabine (2 g/m2/d for 5 d) plus fludarabine (30 mg/m2/d for 5 d) (both agents were dose adjusted for
patients under 12 kg) can also be utilized and is generally well tolerated.

To assess the efficacy of such therapies, clinical response criteria have been defined:

1. White blood count. Complete clinical response defined as WCC less than 20,000/uL; partial clinical response as WCC less than 50% of baseline but with total WCC still greater than 20K.
2. Splenomegaly. CCR normalised spleen; partial clinical response a 25% decrease from initial spleen size.

Potential new method: allele-specific PCR-based strategies to track specific mutations.

Question 24

Potential targeted therapies under investigation for JMML?

Answer 24

Potential therapies include Janus kinase (JAK) inhibitors, based on work demonstrating a pSTAT5 response in JMML cells upon exposure to subtherapeutic concentrations of GM-CSF,43 as well as other signal transduction components of Ras signaling, such as PI3K (phosphoinositide 3-kinase), MEK (MAPK/extracellular-signal-regulated kinase [ERK] kinase), and mTOR (mammalian target of rapamycin) inhibitors.

Question 25

HSCT conditioning regimens in JMML?

Answer 25

Chemotherapy-based conditioning regimens, such as the one published by the EWOG-MDS, utilize busulfan, cytoxan, and melphalan, with reasonable transplant related mortality and morbidity.36,45 The use of total body irradiation is now generally reserved for second transplants, and should be used cautiously in young patients, who may experience substantial late effects. Novel approaches to conditioning regimens are being
developed that may incorporate strategies to further decrease late effects for JMML patients.

Question 26

How quickly should one aim to taper down immunosuppression post HSCT for JMML?

Answer 26

Rapid taper of immunosuppression after HSCT should be initiated as quickly as possible, because a graft-versus-leukemia effect in JMML has been documented

Question 27

Describe traditional and novel methods of detecting relapse post HSCT for JMML?

Answer 27

novel methods of detecting relapse early post-transplant
beyond traditional donor chimerism include sorted cell donor chimerism, quantitative PCR to assess chimerism, as well as allele-specific PCR strategies to detect mutation-specific clones. Such sensitive measurements may facilitate earlier and more rapid withdrawal of immunosuppression or the initiation of novel targeted
therapies. There are minimal data on the utility of donor lymphocyte infusions

Question 28

What is the outlook for JMML patients who relapse post HSCT?

Answer 28

For those JMML patients who relapse after HSCT, there is growing experience with the use of second HSCT, with either the identical donor or an alternative donor. As many as 50% of patients with relapsed JMML can be salvaged with second transplant (Locatelli, unpublished data).

Question 29

In what proportion of JMML patients can a firm molecular diagnosis be made (2010)?

Answer 29

90%

Question 30

For JMML patients with CBL mutations, what serious late sequelae has been recognised?

Answer 30

Tend to get vasculitic disorders. Unclear whether HSCT protects from this and whether this should influence the watch-and-wait vs transplant decision for ‘low risk’ JMML.